To establish the inflammatory cell model, RAW264.7 cells (15 × 104 cells/mL) were stimulated with lipopolysaccharide (1 μg/mL) (LPS, Solarbio, China) following previously reported work, which also confirmed that LPS stimulation could increase the amount of cfDNA [36 ]. After 24 h’ incubation, the stimulated cells were further co-cultured with 50 μg/mL PDA NPs for another 24 h. Finally, the treated cells were used for further researches including live/dead staining, qRT-PCR and immunofluorescent staining. The live/dead staining was implemented using a Calcein- AM/PI assay kit (Sigma, USA). Briefly, PDA NPs treated RAW264.7 cells were incubated with calcein and propidium iodide (PI) for 5 min in the darkness. After PBS buffer washing, the cells were imaged by microscope (Olympus BX53, Tokyo, Japan).
Calcein am pi assay kit
Manufactured by Merck Group
Sourced in United States
The Calcein-AM/PI assay kit is a laboratory tool designed to assess cell viability and cytotoxicity. It utilizes the fluorescent dyes Calcein-AM and Propidium Iodide (PI) to differentiate between live and dead cells. Calcein-AM is a cell-permeant dye that is converted to a green fluorescent compound by esterases in live cells, while PI is a nucleic acid-binding dye that can only enter cells with compromised membranes, indicating cell death.
Automatically generated - may contain errors
Lab products found in correlation
Lipopolysaccharides (lps), by Solarbio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffer saline (pbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified essential medium dmem, by Thermo Fisher Scientific (1 mentions)
Microplate spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Dojindo Laboratories (1 mentions)
3 protocols using calcein am pi assay kit
1
Inflammatory Cell Model with PDA NPs
2
Synthesis and Characterization of IR1061 Dye Compounds
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The chemicals including 1-(4-hydroxyphenyl)ethan-1-one, 4-(diethylamino)benzalde hyde, Potassium hydro-xide (KOH), methanol (MeOH), Nitromethane (MeNO2), 1,2-diethylamine (DCE), ammonium acetate (NH4OAC), n-butanol (n-BuOH), boron (tri) fluoride etherate (BF3Et2O), N, N-Diisopropylethylamine (DIPEA), dichloromethane (DCM), petroleum ether (PE), ethyl acetate (EA), cyclohexane, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phospho ethanolamine-N-PEG2000 (DSPE-PEG2000), methylthiazolyldiphenyl-tetrazolium bro mide (MTT), Calcein-AM/PI assay kit, 4-[(E)-2-[(3Z)-2-chloro-3-[2-(2,6-diphenyl thiopyran-4-ylidene)ethylidene] cyclohexen-1-yl]ethenyl]-2,6 diphenylthiopyrylium, tetrafluorobo-rate (IR1061) were purchased from Sigma-Aldrich and used without further purification. Dulbecco’s modified essential medium (DMEM), Fetal bovine serum (FBS), Streptomycin-penicillin and phosphate buffer saline (PBS) were purchased from Gibco (Life Technologies, Carlsbad, CA, USA). Deionized water (18.2 MΩ·cm) was purified with a water purification system (WP-UP-UV-20) from Sichuan Water Technology Development Co. Ltd. (Chengdu, China). All other chemicals were used as received.
3
Chondrocyte Cytotoxicity Assessment of Ibuprofen Microspheres
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The cell cytotoxicity of Ibu and Ibu-loaded microspheres was detected by using cell counting kit-8 (CCK-8, Dojindo, Japan). Briefly, chondrocytes were seeded in 96-well plates with a density of 8,000 cells per well, and the medium was replaced with fresh culture medium with Ibu (0, 6.25, 12.5, 25, 50, 100, 200, 400 and 800 µg/ml). After incubation for another 48 h, the chondrocytes were rinsed with PBS for three times and replaced with medium containing 10% CCK-8 solution (100 µl). The absorbance was recorded at 450 nm in a microplate spectrophotometer (Thermo Fisher, USA). To obtain the relative cell viability, the absorbance of other groups was compared with the control group. Furthermore, the protective effects of Ibu-loaded microspheres in IL-1β (10 ng/µl, 24 h) induced chondrocytes were also tested by CCK-8 assay. All experiments were repeated in triplicates.
Besides, Live/dead staining was conducted using a Calcein-AM/PI assay kit (Sigma, USA). The chondrocytes were incubated with calcein and propidium iodide (PI) for 5 min in the darkness. After rinsing with PBS, the live/dead cells were imaged by a microscope (Olympus BX53, Tokyo, Japan).
Besides, Live/dead staining was conducted using a Calcein-AM/PI assay kit (Sigma, USA). The chondrocytes were incubated with calcein and propidium iodide (PI) for 5 min in the darkness. After rinsing with PBS, the live/dead cells were imaged by a microscope (Olympus BX53, Tokyo, Japan).
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